Control of airway surface liquid (ASL) volume is vital for pulmonary defense against inhaled pathogens/toxicants. Deficits in ASL volume produce airways obstruction and airways infection, reflecting the absence of periciliary liquid (PCL) volume and adhesion of dehydrated mucus to airway surfaces. Much is known about the ion transport processes that control transepithelial ion fluxes, but there are little or no data describing how these processes are coordinately regulated to adjust the mass of salt and, hence, water on airway surfaces in the ranges required for health. Studies of patients with genetic lung diseases, e.g., cystic fibrosis, have suggested that regulation of both the CFTR and ENaC channels are vital for this process. More recently, a number of clues have suggested a role for nucleotides (NTs) and nucleosides (NSs) in ASL in regulating the balance between Na+ absorption and Cl- secretion to generate ASL volume homeostasis. Indeed, we hypothesize that 1) ASL [NT+NS]s are so critical for ASL volume regulation that in their absence, airway epithelia revert to a purely Na+-absorbing state and deplete all ASL from airway surfaces;and 2) the volume of ASL is proportional to the rate of ATP release (JATP) onto airway surfaces. To test these hypotheses and generate a comprehensive description of ASL volume homeostasis, we propose three Specific Aims: 1) Aim 1 - measure JATP and extracellular NT+NS metabolism to develop a mathematical model that will integrate ASL NT+NS concentrations with a biophysical model of ion transport to describe the regulation of ASL volume homeostasis;2) Aim 2 - test in human bronchial epithelial (HBE) cultures the requirement for NTs and NSs in the acute regulation of ASL volume homeostasis and the mechanisms that mediate these regulatory processes;and 3) Aim 3 - test the requirement for NT+NS in controlling ASL volume in mutant mouse models in vivo. Relevance to Public Health: Accurate quantitative knowledge of the factors that control ASL homeostasis, i.e., the 'hydration'of airway surfaces, will aid in elucidation of the pathogenesis of major human airways diseases, e.g., COPD, CF, and asthma, and will provide insights into novel therapeutic mechanisms to hydrate airway surfaces and hence, restore normal host defense.